| Glycosaminoglycans (GAGs) are a family of highly sulfated, linear polysaccharides that display a wide array of biological activities. Heparin, the most prominent GAG, exhibits an array of important biological activities through its interaction with coagulation proteins, growth factors, and chemokines. A deeper understanding of the biological functions of GAGs requires new analytical methods for the elucidation of their fine structures. A number of advances have been made for the separation of GAGs and GAG-derived oligosaccharides. New derivatization methods, chromatography and electrophoresis-based separations and NMR and mass spectrometry methods are being applied to solve complex oligosaccharide structures. Combining separation and mass spectral techniques is also critical.; The focus of the current work was on the improvement of an analytical method for structural study to better understand heparin's structure. A reversed-phase ion-pairing high performance liquid chromatography (RPIP-HPLC) method for the separation of a complex mixture of heparin-derived oligosaccharides was developed by a stepwise optimization of the mobile phase. The resolution of more than 30 oligosaccharide components was obtained, under optimized conditions, in an analysis time of less than 30 minutes. This represents the first reversed-phase HPLC method that can separate a complex mixture of both small and large sulfated oligosaccharides in a single chromatographic step.; To our knowledge, a generally useful separation method is still not available for large and highly sulfated oligosaccharides, which enables coupling with mass spectrometric detection without significant difficulties. In this study we reported the use of RPIP-HPLC separation coupled to negative-ion electrospray mass spectrometry for the analyses of the heparin oligosaccharides. The chromatographic separations are made possible through the use of a volatile ion-pairing reagent, tributylammonium acetate, together with ammonium acetate as an additive to the mobile phase, and linear gradient elution of acetonitrile. Highly sensitive and easily interpretable spectra were obtained through post-column addition of tributylamine in acetonitrile. High-resolution mass spectrometry afforded elemental composition of many known and previously unknown heparin-derived oligosaccharides. The structural identification of these oligosaccharides provided sequence from a reading frame that begins at the non-reducing terminus of the heparin chain. |
